Digital DNA typing

ABSTRACT

To sequence DNA automatically, fluorescently marked DNA are electrophoresed in a plurality of channels through a gel electrophoresis slab; wherein the DNA samples are resolved in accordance with the size of DNA fragments in the gel electrophoresis slab into fluorescently marked DNA bands. The separated samples are scanned photoelectrically with a laser and a sensor, wherein the laser scans with scanning light at a scanning light frequency within the absorbance spectrum of said fluorescently marked DNA samples and light is sensed at the emission frequency of the marked DNA. The light is modulated from said laser at a predetermined modulation frequency and fluorescent light emitted by said DNA bands at said modulation frequency is detected, whereby background noise from the medium through which the light is transmitted is discriminated against.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation, of application Ser. No. 07/959,262,filed Oct. 9, 1992, now abandoned which is a continuation-in-partapplication of Ser. No. 07/570,503 filed Aug. 21, 1990 now U.S. Pat. No.5,207,880, which is a continuation-in-part application of Ser. No.07/078,279 filed Jul. 27, 1987 now abandoned, which is a division ofU.S. application Ser. No. 594,676 filed Oct. 10, 1990 for DNA SEQUENCINGfiled by Middendorf et al. on Oct. 10, 1990, and assigned to the sameassignee as this application, now U.S. Pat. No. 5,030,345.

BACKGROUND OF THE INVENTION

This invention relates to the sequencing of DNA strands.

In one class of techniques for sequencing DNA, identical strands of DNAare marked. The strands are separated into four aliquots. The strands ina given aliquot are either individually cleaved at or synthesized to anybase belonging to only one of the four base types, which are adenine,guanine, cytosine and thymine (hereinafter A, G, C and T). The adenine-,guanine-, cytosine- and thymine-terminated strands are thenelectrophoresed for separation. The rate of electrophoresis indicatesthe DNA sequence.

In a prior art sequencing technique of this class, the DNA strands aremarked with a radioactive marker, cleaved at any base belonging to abase type unique to the aliquot in which the strands are contained, andafter being separated by electrophoresis, film is exposed to the gel anddeveloped to indicate the sequence of the bands. The range of lengthsand resolution of this type of static detection is limited by the sizeof the apparatus.

In another prior art sequencing technique of this class, single strandsare synthesized to any base belonging to a base type unique to thealiquot in which the strands are contained, The strands are markedradioactively for later detection.

It is also known in the prior art to use fluorescent markers for markingproteins and to pulse the fluorescent markers with light to receive anindication of the presence of a particular protein from thefluorescence.

The prior art techniques for DNA sequencing have several disadvantagessuch as: (1) they are relatively slow; (2) they are at least partlymanual; and (3) they are limited to relatively short strands of DNA.

In a technique related to DNA sequencing, used to identify restrictionfragment length polymorphisms (RFLPs), DNA strands, cut by restrictionenzymes, are separated into bands by electrophoresis and moved at rightangles onto a blotting matrix containing radioactively labelled probesfor identification. This procedure has the same disadvantages as theprior art sequencing procedures.

Digital DNA Typing using radioactivity labeling and transfer by blottingis known in the prior art from Jeffreys, A., A. MacLeod, K. Tamaki, D.Neil, and D Monckton. Minisatellite repeat coding as a digital approachto DNA typing. Nature 354:204-209, 1991. This method of digital typinghas the disadvantages of requiring radioactivity, being time consumingand limited on the number of DNA bases that can be considered.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the invention to provide a noveltechnique for DNA sequencing.

It is a still further object of the invention to provide novelapparatuses and methods for sequencing relatively large fragments ofDNA.

It is a still further object of the invention to provide novelapparatuses and methods for sequencing DNA fragments of 100 bases ormore.

It is a still further object of the invention to provide a technique forcontinuous sequencing of DNA.

It is a still further object of the invention to continuously sequenceDNA without the spatial limitations of range of lengths and resolution.

It is a still further object of the invention to provide a noveltechnique for continuously sequencing DNA using fluorescent detection.

It is a still further object of the invention to provide a noveltechnique for DNA sequencing using a fluorescent marker fastened to theDNA, or the inherent fluorescence of the DNA itself.

It is a still further object of the invention to provide a noveltechnique for continuously sequencing DNA marked with fluorescence whichmore clearly distinguishes marked DNA fragments from backgroundfluorescence.

It is a still further object of the invention to provide a noveltechnique for continuously sequencing DNA using radioactive detection.

It is a still further object of the invention to provide a novel methodof digital DNA typing.

It is a still further object of the invention to provide a technique fordigital DNA typing without requiring the use of radioactive material.

It is a still further object of the invention to provide a technique fordigital DNA typing using continuous on line optical DNA sequencing.

In accordance with the above and further objects of the invention,strands of DNA are continuously electrophoresed and identified for: (1)DNA sequencing; and (2) analysis of strands varying in length, asprepared by such techniques as restriction enzyme cutting or polymerasechain reaction (PCR), and marked by direct labelling of fluorescentmarkers to the strands or by fluorescently labelled probes hybridized tothe separated strands. The strands are fluorescently marked and thelight emitted during a scan is detected and correlated. The gel andvoltage are selected so that strands being electrophoresed near theterminal end of the gel channel are fully resolved prior to theresolution of longer strands which are at the entrance end of the gelchannel, and so on, in a continuous process over a period of time.

One embodiment of apparatus for sequencing DNA includes at least fourelectrophoresis channels, each adapted to receive fluorescently labeledDNA strands, having at one end a base of a given type. Each of thechannels has a gel path and electrical field across it identical in itscharacteristics to the gel path of the other channels and electricalfields across the other channels.

To provide marking in one embodiment, a fluorescent marker is attachedto the DNA fragments prior to their being electrophoresed into the gel.In another embodiment, probes are used to combine or hybridize with theDNA strands, with detection accomplished by detecting a fluorescentmarker that is chemically attached to the probe. In the preferredembodiment, the marker is a dye that fluoresces in the infrared or nearinfrared region.

In another embodiment, biotin is attached to the DNA fragments prior totheir being electrophoresed into the gel and at the terminal end of thegel, avidin is applied to the strands to further mark the strandsindividually while maintaining the strands in each channel separate fromthe strands in other channels. The avidin is pre-marked with multiplefluorescent molecules and therefore provides multiple fluorescentmarkers for each separated strand.

In other embodiments, after separation: (1) the inherent fluorescence ofDNA is used as a suitable detection mechanism, so that it is notnecessary to mark one end of the strands with biotin nor mark them witha fluorescent marker nor attach fluorescently labelled primers; or (2)radioactive markers attached directly to the DNA are used as a suitabledetection mechanism.

The gel electrophoresis may be provided in conventional gel slabs. Inone embodiment, there is a different input section for each of fourchannels that are for a corresponding one of the A, G, T and C strands.

As alternatives to a gel slab: (1) four capillary tubes may be used withgel in them to allow higher voltages per unit length of the gel withoutexcess heating, thereby allowing faster separation; (2) open capillarytubes may be used and thus avoid the need for gel and make the cleaningmore convenient; (3) high performance liquid chromatography (HPLC)columns such as ion-exchange columns or reverse phase columns may beused in conjunction with pressure instead of voltage for separating thestrands; or (4) fewer or more than four channels may be used with orwithout marking that separates the DNA strands.

The strands are detected during electrophoresis either in the gel orafter leaving the gel by scanning back and forth across the gel at afixed distance from the entrance end of the gel or by moving the strandsby bulk flow, such as through the use of a moving blotting membrane pasta detector or detectors. Means are provided for detecting the bandsindividually in each channel in accordance with their mobility in thegel to indicate the sequence of the A, G, C and T strands of differentlengths. Advantageously, an additional channel may be utilized as acalibration channel through the electorphoresis of DNA strands of known,but different lengths. These DNA strands are also marked and therebyindicate a time base.

The scanning apparatus includes a light source, such as a laser or arclamp or other suitable source that emits light in the optimum absorptionspectrum of the marker. The light may be split by the use of fiber. Inthe preferred embodiment, the light source is a diode laser that scansthe channels with infrared light having a wave length that matches theabsorbance region of the marker. The detector includes a light sensorwhich is preferably an avalanche photodiode sensitive to the nearinfrared light emission of the marker. It may include a filtering systemhaving a band pass suitable for passing selectively the optimum emissionof the fluorescent marker to the light sensor.

The photodiode, photomultiplier or other light detector selectivelydetects the fluorescence using techniques which enhance the signal/noiseratio. One technique is to modulate the laser source by pulsing theelectrical current driving the laser, with detection through a lock-inamplifier.

Another technique is the use of laser pulses which are less than fivenanoseconds time duration, with detection in a time window. The lengthof such window and its delay from the pulse are optimized todiscriminate against background fluorescence. Correlation with thechannel in which the fluorescent light is detected with the time ofdetection indicates: (1) if the type of base termination or nucleotidecleavage is A, G, C or T; and (2) the time sequence of separation ofeach strand in each channel of the electrophoresis gel column toindicate the overall sequence of strands.

To use the apparatus to sequence DNA strands, identical DNA strands arenormally formed of a length greater than 100 bases. In one embodiment,the strands are marked by a suitable marker at one end. The strands aredivided into four aliquots and the strands within each aliquot arecleaved at any base belonging to a specific base type. In anotherembodiment, strands are synthesized to any base belonging to a specificbase type. These four aliquots are then electrophoresed throughidentical channels to separate strands so that the shorter strands areresolved towards the end of the gel prior to resolution of the longerstrands, which still are near the entrance end of the gel. This occursin a continuous process so a substantial number of different lengthstrands may be resolved in a relatively short gel. This methodologytakes advantage of time-resolved bands, as opposed to the limitations ofspatial-resolved bands.

The gel size, electric field and DNA mobilities are such that the moremobile bands are fully resolved while the less mobile bands are yetunresolved in a continuous process such that at least ten percent of thebands have been resolved by electrophoresis in the gel while the lessmobile bands which are near the entrance end of the gel are not fullyresolved. These less mobile bands become resolved little by little overtime in a continuous fashion without interruption of the movement ofthese bands through the gel.

The markers are detected by transmitting light to the markers. In thepreferred embodiment, light is transmitted to a fluorescent marked DNAstrand. In the latter case, the fluorescent light is detected either bymodulating the light source and detecting using lock-in techniques ordetecting during a time period in which the markers' fluorescence hasnot yet decayed to an insignificant amount but the backgroundfluorescence has. The detection is made in a wavelength band includingthe high emission spectrum of the fluorescent marker.

For the gated window technique, the light is transmitted from pulsedlasers in approximately three nanosecond pulses. Readings are takenwithin a window period, after an initial delay, and both period anddelay are optimized for best results. In another embodiment, radioactivemarked strands, after being separated, excite a phosphor orscintillation liquid whereby detection of the presence of the strands isaccomplished by an appropriate photodetector.

To provide an improved DNA typing procedure, a prior art radioactivetechnique for digital typing of DNA is modified for use with an opticalon-line scanner. For this purpose, the technique described in theaforementioned article, Jeffreys, A., A. MacLeod, K. Tamaki, D. Neil,and D. Monckton; Mini Satellite Repeat Coding As A Digital Approach ToDNA Typing; Nature 354:204-209, 1991, is modified and used. Themodifications include using a 32D probe which has an extension of thesame sequence as another primer that has an infrared dye attached;sequencing the fragments of different length (sequential typing)according to the above procedures using a denaturing acrylamide gel asthe separation medium; and collecting data in a continuous fashion asdescribed above.

Generally, a DNA locus that varies in sequence from one individual toanother is amplified using PCR and processed to be sequentially typed bythe laser sequencer as described in this application. This can beaccomplished by determining the sequential typing information and usingthe sequential typing information as a code to identify the DNA.Preferably a portion of the locus is amplified or a correspondingsection of bases for each of several localities such as the definedterminal bases of multiple tandem repeats are amplified by PCR to form aplurality of identical strands. The strands are sequentially typed asdescribed above. The sequential typing information is then consideredthe identification code. The typing information is designated in binaryfashion for information coded by units having two differing basesequence structures or in ternary fashion for information coded by unitshaving three differing base sequence structures or the like. The digitalpatterns are compared so that the same code indicates the sameindividuals.

From the above summary, it can be understood that the sequencingtechniques of this invention have several advantages, such as: (1) theytake advantage of resolution over time, as opposed to space; (2) theyare continuous; (3) they are automatic; (4) they are capable ofsequencing or identifying markers in relatively long strands includingstrands of more than 100 bases; and (5) they are relatively economicaland easy to use.

SUMMARY OF THE DRAWINGS

The above noted and other features of the invention will be betterunderstood from the following detailed description when considered withreference to the accompanying drawings in which:

FIG. 1 is a block diagram of an embodiment of the invention;

FIG. 2 is a block diagram of another embodiment of the invention;

FIG. 3 is a simplified perspective view of a portion of the embodimentof FIGS. 1 and 2;

FIG. 4 is a block diagram of a portion of the embodiment of FIGS. 1 and2;

FIG. 5 is a schematic circuit diagram of a protion of the embodiment ofFIGS. 1 and 2;

FIG. 6 is a schematic circuit diagram of a portion of the schematicdiagram of FIG. 5;

FIG. 7 is a block diagram of still another embodiment of the invention;

FIG. 8 is a block diagram of still another embodiment of the invention;

FIG. 9 is a block diagram of another embodiment of the invention;

FIG. 10 is a perspective view of a portion of the embodiment of FIG. 9;

FIG. 11 is a sectional view taken through lines 11--11 of FIG. 10;

FIG. 12 is a sectional view of a portion of FIG. 10 taken through lines12--12;

FIG. 13 is an exploded perspective view of a portion of the embodimentof FIG. 11;

FIG. 14 is an enlarged view, partly broken away, of a portion of theembodiment of FIG. 11; and

FIG. 15 is a block diagram of a circuit that may be used forcoordination of a sensor, scanner drive and laser used in the embodimentof FIG. 9.

DETAILED DESCRIPTION

In FIG. 1, there is shown a block diagram of one embodiment of a DNAsequencing system 10 having a biotin labeling system 11, a DNA cleavagesystem 12, a separating system 14, a detection and processing system 16and a source of standard length DNA 18. The biotin from any suitablecommercial source is attached to the cloned strands of more than 100bases in a container as indicated at 11. The biotin preparation must besufficient to mark at least one end of a substantial proportion of theDNA fragments with the biotin in a manner known in the art.

Although biotin has been selected as one marker which may be combinedlater with a larger fluorescently marked molecule such as avidin, othermarkers may be used. Such markers may be fluorescent and therefore donot require the subsequent combination with a larger fluorescentlymarked molecule such as avidin. In addition, multiple fluorescentmarkers may be attached to the DNA fragments. They must be of such asize and have such chemical characteristics to not obscure the normaldifferences in the mobilities between the different fragments due tocleavages at different ones of the adenine, guanine, cytosine andthymine bases and be able to be easily detected.

The DNA cleavage system 12 communicates in four paths and the source ofstandard length DNA 18 communicates in one path within the separatingsystem 14 to permit passage of DNA fragments and standard fragmentsthereto in separate paths. The separating system 14, which sequencesstrands by separation, communicates with the detection and processingsystem 16 which analyzes the fragments by comparison with each other andthe standard from the source of standard length DNA 18 to deriveinformation about the DNA sequence of the original fragments.

The DNA cleavage system 12 includes four sources 20A, 20G, 20C and 20Tof fragments of the same cloned DNA strand. This DNA strand is normallygreater than 100 bases in length and is then further cleaved by chemicaltreatment to provide different lengths of fragments in each of fourcontainers 20A, 20G, 20C and 20T.

In one embodiment, the container 20A contains fragments of DNA strandsrandomly cleaved by a chemical treatment for A, the container 20Gcontains fragments of DNA strands randomly cleaved by a chemicaltreatment for G; container 20C contains fragments of DNA strandsrandomly cleaved by a chemical treatment for C; and container 20Tcontains fragments of DNA strands randomly cleaved by a chemicaltreatment for T. Thus, identical fragments in each container have beencleaved at different bases of a given base type by the appropriatechemical treatment.

The fragments in the containers are respectively referred to as A-DNAfragments, G-DNA fragments, C-DNA fragments and T-DNA fragments from thecontainers 20A, 20G, 20C and 20T, respectively. These fragments areflowed from the containers 20A, 20G, 20C and 20T through correspondingones of the conduits 22A, 22G, 22C and 22T into contact with theseparating system 14. In the preferred embodiment, conduits 22A, 22G,22C and 22T represent the pipetting of samples from containers 20A, 20G,20C and 20T into the separately system 14.

The source of standard length DNA 18 includes a source of reference DNAfragments of known but different lengths which are flowed through aconduit 22S, such conduit including, but not limited to a pipettingoperation to the separating system 14. These reference fragments haveknown lengths and therefore their time of movement through theseparating system 14 forms a clock source or timing source as explainedhereinafter. The cloned strands of 100 bases may be marked with biotinor with one or more fluorescent molecules before being divided into fourbatches or they may be marked instead after dividing into four batches,but before the selected chemical treatment.

The separating system 14 includes five electrophoresis channels 26S,26A, 26G, 26C and 26T. The electrophoresis channels 26S, 26A, 26G, 26Cand 26T include in the preferred embodiment, gel electrophoresisapparatus with each path length of gel being identical and having thesame field applied across it to move samples continuously through fivechannels. The gels and fields are selected to provide a mobility to DNAstrands that does not differ from channel to channel by more than 5% invelocity. In addition, the field may be varied over time to enhance thespeed of larger molecules after smaller molecules have been detected, aswell as to adjust the velocities in each channel based on feedback fromthe clock channel to compensate for differences in each channel suchthat the mobilities in each channel are within the accuracy required tomaintain synchronism among the channels.

Preferably the gels are of the same materials, chemical derivatives andlengths and the electric fields are within 5% of the intermediates ofeach other in each channel. However, more than one reference channel canbe used such that a reference channel is adjacent to a sample channel inorder to minimize the requirements for uniformity of DNA movement in thegel for all channels.

The electrophoresis channel 26S receives fragments of known length DNAmarked with biotin or with one or more fluorescent molecules and movesthem through the gel. Similarly, each of the electrophoresis channels26A, 26G, 26C and 26T receives labeled fragments from the cleavagesystem 12 and moves them in sequence through the sample electrophoresischannels, with each being moved in accordance with its mobility under afield identical to that of the reference electrophoresis channel 26S.

To provide information concerning the DNA sequence, the detection andprocessing system 16 includes five avidin sources 30S, 30A, 30G, 30C and30T; five detection systems 32S, 32A, 32G, 32C and 32T and a correlationsystem 34. Each of the avidin sources 30S, 30A, 30G, 30C and 30T isconnected to the detecting systems 32S, 32A, 32G, 32C and 32T. Each ofthe outputs from corresponding ones of the electrophoresis channels 26S,26A, 26G, 26C and 26T within the separating system 14 is connected to acorresponding one of the detection systems 32S, 32A, 32G, 32C and 32T.

In the detection system, avidin with fluorescent markers attached andDNA fragments are combined to provide avidin marked DNA fragments withfluorescent markers attached to the avidin to a sample volume within thedetection system for the detection of bands indicating the presence orabsence of the fragments, which over time relates to their length. Inthe embodiment where the DNA strands are marked with one or morefluorescent markers rather than with biotin, it is not necessary tocombine such DNA strands with avidin, and the DNA strands are moveddirectly into the detection system.

The output from each of the detection systems 32S, 32A, 32G, 32C and 32Tare electrically connected through conductors to the correlation system34 which may be a microprocessor system for correlating the informationfrom each of the detection systems to provide information concerning theDNA sequence.

The avidin sources 30S, 30A, 30G, 30C and 30T each contain avidinpurchased from known suppliers, with each avidin molecule in thepreferred embodiment combined with three fluorescein molecules. Theavidin sources are arranged to contact the DNA fragments and may becombined with the biotin-labelled DNA strands after such strands havebeen electrophoresed onto a moving blotting membrane.

The detection systems each include an optical system for detecting thepresence or absence of bands and converting the detection of them toelectrical signals which are applied electrically to the correlationsystem 34 indicating the sequence of the fragments with respect to boththe standard fragments from the source of standard length DNA 18 and theA, G, C and T fragments from the containers 20A, 20G, 20C and 20T,respectively.

In FIG. 2, there is shown a simplified block diagram of anotherembodiment of DNA sequencing apparatus A10. This apparatus is similar tothe DNA sequencing apparatus 10 of FIG. 1 and the components areidentified in a similar manner with the reference numbers being prefixedby the letter A.

In this embodiment, instead of the containers for DNA and chemicaltreatment for A, G, C and T of the embodiment of DNA sequencing system10 shown at 20A, 20G, 20C and 20T in FIG. 1, the DNA sequencingapparatus A10 includes containers for treatment of the DNA in accordancewith the method of Sanger described by F. Sanger, S. Nicklen and A. R.Coulson, "DNA Sequencing with Chain-Terminating Inhibiters", Proceedingsof the National Academy of Science, USA, Vol. 74, No. 12, 5463-5467,1977, indicated in the embodiment A10 of FIG. 2 at A20A, A20G, A20C andA20T shown as a group generally at A12.

In this method, the strands are separated and used as templates tosynthesize DNA with synthesis terminating at given base types A, G, C orT in a random manner so as to obtain a plurality of different molecularweight strands. The limited synthesis is obtained by using nucleotideswhich terminate synthesis and is performed in separate containers, oneof which has the special A nucleotide, another the special G nucleotide,another the special C nucleotide and another the special T nucleotide.These special nucleotides may be dideoxy nucleotides or markednucleotides, both of which would terminate synthesis. Such markednucleotides may be fluorescent. Each of the four batches will beterminated at a different one of the types of bases A, G, C and Trandomly. This synthesis takes place in containers A20A, A20G, A20C andA20T.

In the preferred embodiment of FIG. 2, the template fragments arehybridized with a DNA primer having one or more fluorescent markersattached to it as shown at A11 before being applied to the channelsindicated at A12 in FIG. 2. The design of fluorescently-labelled primerstakes advantage of the process of designing small DNA fragments known asoligonucleotides. This process is described in the scientific and patentliterature, such as for example U.S. Pat. No. 4,415,732, the disclosureof which is incorporated herein.

The synthesized strands, labelled by the fluorescently-marked primers,are electrophoresced in channels A26A, A26G, A26C and A26T. After theelectrophoresis, the synthesized DNA fragments with the attachedfluorescently-labelled primer are detected by the detection system usinga wavelength of light appropriate to the emission spectrum of thefluorescent markers.

In FIG. 3, there is shown a separating system 14 which includes a slabof gel 27 as known in the art with five sample dispensing tubesindicated generally at 29A terminating in aligned slots 51 in the gel 27on one end, with such slots in contact with a negative potential bufferwell 29 having a negative electrode 47A, and five exit tubes at theother end located at 31A terminating in apertures in the gel 27, as wellas a positive potential buffer well 31 having a positive electrode 53A.

The material to be electrophoresed is inserted into dispensing tubes 29Aand due to the field across the gel 27 moves from top to bottom in thegel and into the appropriate corresponding exit tubes of the group 31A.The gel slab 27 has glass plates 27A and 27B on either side to confinethe sample and gel. Buffer fluid from the buffer well 31 is pumped atright angles to the gel 27 from a source at 57 by pumps connected toexit tubes 31A to pull fluid there through. The buffer fluid picks upany DNA that is electrophoresed into the exit tubes 31A and makes itsway to sensing equipment to be described hereinafter or to providecommunication with other gel slabs for futher electrophoresis of the DNAstrands being electrophoresed from the slab 27.

In FIG. 4, there is shown a block diagram of the detection system 32A.The detection systems 32S, 32G, 32C and 32T (not shown in FIG. 4) aresubstantially identical to the detection system 32A and so only thesystem 32A will be described in detail herein. The detection system 32Aincludes an electrophoresis channel 42 (which may be a continuation ofelectrophoresis channel 26A as indicated in FIG. 1), a sample volume 43(which may be part of electrophoresis channel 42), a light source 44 andan optical detection system 46.

In one embodiment, avidin marked with fluorescein influorescenated-avidin source 40 is brought into contact with the gelwhich receives A type terminated strands from electrophoresis channel26A (FIG. 1) on conduit 48 and such fluorescein-labelled avidin isattached to the biotin-labelled DNA fragments. The electrophoresischannel 42 may be a continuation of electrophoresis channel 26A forcontinuous electrophoresing. After the fluorescein-labelled avidin isattached to the biotin-labelled DNA in the electrophoresis channel 42,the complex molecule is moved into the sample volume 43.

The sample volume 43 is irradiated by the light source 44. Light fromthe light source 44 is detected and converted to electrical signals bythe optical detection system 46 for application through a conductor 50Ato the correlation system 34 (FIG. 1). In one embodiment, thefluorescenated-avidin source 40 contains a fluorescent marker having aperiod of fluorescence sufficiently long compared to backgroundfluorescence of the gel and associated materials to permit significantseparation of the signal from the fluorescence.

The light source 44 includes a pulsed light source 52 and a modulator54. The pulsed light source 52 is selected to emit light within theabsorbance spectrum of the fluorescent marker. In one embodiment, themodulator 54 controls the pulsed light source 52 to select intervalsbetween pulses, the intervals being provided to permit the decay offluorescent light from the background fluorescent material, during whichtime the fluorescent light from the fluorescent markers is measured.

These time periods between pulses are sufficiently long to emcompass theentire delay period. This is done because the delay period of theattached fluorescent marker is relatively long compared to backgroundnoise fluorescence and so a period of time may pass before themeasurement is made by the optical detection system 46. Typically, thepulse of light has a duration of approximately three nanoseconds and thebackground fluorescence decay lasts for approximately ten nanosecondswhile the fluorescent marker has a decay lifetime of 100 nanoseconds.

Typically, the optical detection system 46 begins reading atapproximately 50 nanoseconds after the initiation of the excitationpulse from a laser and continues for approximately 150 nanoseconds until200 nanoseconds after the initiation of the three nanosecond pulse.Although in this embodiment, a pulsed laser light source 52 is utilized,a broad band light source combined with filters or a monochrometer maybe utilized to provide the narrow band in the absorption spectrum of themarker.

Another embodiment uses an electro-optic modulator which modulates acontinuous light source at a frequency typically at 10 khz, withessentially 100% depth of modulation and 50% duty cycle. A pulsegenerator provides a signal both to the modulator through a driver andto a lock-in amplifier as a reference signal. Another embodimentprovides modulation of a laser diode light source through the pulsing ofthe drive current to the laser diode. Modulation may typically be atfrequencies between 100 and 15,000 hz with a 50% duty cycle. A lock-inamplifier is used to synchronously demodulate the fluorescent signal.Another embodiment uses a spinning chopping wheel to modulate acontinuous light source. Because the background fluorescence signal fromglass (soda lime, borosilicate, quartz, etc.) has a larger time constantthan that of the fluorescent marker, temperal discrimination isaccomplished by modulating the light source. Still another embodimentuses a continuous light source with no modulation.

To detect the bands in the electrophoresis gel of the electrophoresischannel 42 indicating particular DNA fragments, the optical detectionsystem 46 includes certain viewing optics 60, a filter 62, and anoptical detection system 64. The filter 62 selects the wavelength oflight transmitted through it by the viewing optics 60 which focuses thelight onto the optical detection system 64. The optical detection system64 is electrically connected to the modulator 54. The signal onconductor 50A indicates the presence or absence of a band of DNAfragments in the sample volume 43.

The filter 62 in this embodiment includes an interference filter havinga pass band corresponding to the high emission spectrum of thefluorescent marker. Such filters are known in the art and may bepurchased from commercial sources with bands to correspond to commonemission bands of fluorescent markers. In addition, there may belong-wavelength-passing intereference filters and/or colored glassfilters. Another embodiment uses a monochrometer instead of a filter.

The viewing optics 60 consists of a lens system positioned injuxtaposition with filter 62 to focus light onto the optical detectionsystem 64. It may be any conventional optical system, and the opticaldetection system 64 should include a semiconductor detector or aphotomultiplier tube, such as the Model R928 made by Hamamatsu, Japan.

In the first embodiment, the output of the photomultiplier orsemiconductor detector is gated in response to the signals from themodulator 54 to occur after a time delay after each pulse from thepulsed laser light source 52. For example, a time delay may be includedbefore the electrical signal is applied to an amplifier and thus providean electrical signal to the conductor 50A or to an amplifier, the outputof which is electrically connected to the conductor 50A. In thepreferred embodiment, the time delay is 50 microseconds and the gate oramplifier is maintained opened by a monostable multivibrator forapproximately 150 nanoseconds. In a second embodiment, the square waveoutput of a modulator is used as a reference for the signal from thedetector, with a lock-in amplifier providing synchronous demodulation.In a third embodiment, no modulation is performed.

In FIG. 5, there is shown a block diagram of the correlation system 34having a standard channel input circuit 70S, a gating system 72, adecoder 74, a memory 76 and a read-out system 78. An OR gate 74S iselectrically connected to: (1) the standard channel input circuit 70S;(2) other channels 70A, 70G, 70C and 70T; and (3) the gating system 72.The gating system 72 receives channel input signals from each of thechannels 70A, 70G, 70C, and 70T similar to that of channel 70S.

The OR gate 74S is electrically connected to the memory 76 whichreceives signals from decoder 74 indicating the presence of DNAfragments in the particular one of the nucleic acid bases or in thestandard channel. The memory 76 is electrically connected to theread-out system 78 to print out the sequence.

The standard channel input circuit 70S includes a pulse shaper 82S, abinary counter 84S, and a latch 86S, with the input of the pulse shaper82S being electrically connected to a conductor 50S and its output beingconnected to OR gate 74S. The output of the binary counter 84S isconnected to the latch 86S to provide a time increment signal to thelatch 86S, the output of which is applied to one of the inputs of memory76 when triggered by a signal from OR gate 74S. The conductor 50Scorresponds to conductors 50A, 50G, 50C and 50T except that conductor50S is the output for the standard clock channel rather than foradenine, guanine, cytosine or thymine.

The latch 86S and the decoder 74 are pulsed by a signal from the OR gate74S to write into the memory 76 for recording with a distinctive signalindicating a clock timing pulse which is later printed to indicate thetime that particular DNA segments have been received and detected in thedetection system 32A, 32G, 32C and 32T (FIG. 1). The binary counter 84Sreceives clock pulses from clock 80 to which it is connected and thuscontains a binary signal representing time for application to the latch86S.

The gating system 72 includes a decoder 74 which is electricallyconnected to four inputs from channels 70A, 70G, 70C and 70Trespectively, for receiving signals indicating the presence of types A,G, C, and T fragments as they appear on input conductors 50A, 50G, 50Cand 50T. The signals on conductors 50A, 50G, 50C and 50T are eachapplied to respective ones of the pulse shapers 82A, 82G, 82C and 82T,the outputs of which are electrically connected through correspondingones of the conductors 92A, 92G, 92C, and 92T to different inputs of thedecoder 74 and to inputs of the OR gate 74S, so that the decoder 74receives signals indicating the presence of a DNA fragment forapplication to the memory 76 upon receiving a signal on conductor 90Sfrom the OR gate 74S. The OR gate 74S applies such a signal whenreceiving a signal from any one of the channels 70S, 70A, 70G, 70C, and70T, so that the memory 76 receives clock timing signals and signalsindicating DNA for reading to the readout system 78. The output of thedecoder 74 is electrically connected to the memory 76 through aconductor 100.

In FIG. 6, there is shown a schematic circuit diagram of the decoder 74having an OR gate 102 and a plurality of coding channels 74A, 74G, 74Cand 74T to respectively indicate fragments terminating with the bases,adenine, guanine, cytosine and thymine respectively.

The coding channel 74A includes AND gate 106, having its inputselectrically connected to conductor 92A and 90S to receive on conductor90S a clock signal from the OR gate 74S (FIG. 5) and on its other inputa signal indicating the presence of an adenine terminated fragment onconductor 92A.

Channel 74G includes AND gate 108, AND gate 110 and delay line 112.Conductor 92G indicating a guanine terminated strand is electricallyconnected to the inputs of AND gate 108 and 110. The output of AND gate108 is connected to one of the inputs of OR gate 102 and the output ofAND gate 110 is electrically connected through delay line 112 to theinput of OR gate 102 to provide two pulses in succession to OR gate 102.Thus, channel 74A applies one out pulse from the output of AND gate 106to one of the inputs of OR gate 102, whereas channel 74G applies twopulses. In either case, the sequence of pulses indicates the presence ofa particular one of the types of DNA fragments A or G.

Similarly, the channel 74C includes AND gates 114, 116 and 118, eachhaving one of its two inputs electrically connected to conductor 92C and90S and the channel 74T includes AND gates 120, 122, 124 and 126, eachhaving one of its inputs electrically connected to conductor 92T and theother connected to conductor 90S. The output from AND gate 114 iselectrically connected to an input of OR gate 102, the output of ANDgate 116 is electrically connected through a delay 128 to the input ofOR gate 102, and the output of AND gate 118 is electrically connectedthrough a delay 130 longer than the delay 128 to an input of the OR gate102. With this arrangement, the presence of a DNA strand terminatingwith cytosine results in three pulses to the OR gate 102.

The output of AND gate 120 is electrically connected to an input of theOR gate 102, the output of the AND gate 122 is electrically connectedthrough a delay 132 to an input of the OR gate 102, the output of ANDgate 124 is electrically connected through a delay 134 longer than thedelay 132 to an input of the OR gate 102 and the output of AND gate 126is electrically connected through a delay 136 longer than the delay 134to an input of the OR gate 102. In this manner, the presence of athymine-terminated fragment results in four signals in series to theinputs of OR gate 102. The output terminal of OR gate 102 is applied tothe output conductor 100 so as to provide a coded signal indicating thepresence of a particular DNA group to the memory 76 (FIG. 5) coordinatedwith a clock timing signal.

In FIG. 7, there is shown a simplified block diagram of anotherembodiment of DNA sequencing apparatus B10. This apparatus is similar tothe DNA sequencing apparatus 10 of FIG. 1 and the components areidentified in a similar manner with the reference numbers being prefixedby the letter B. However, the strands of DNA are labeled in labelingcontainer B11 with one or more fluorescent molecules. Containers fortreatment of the strands to form fragments to A, G, C or T terminationsin different containers are indicated in the embodiments B10 of FIG. 7at B20A, B20G, B20C and B20T shown as a group generally at B12.

In this method, single-stranded DNA are marked with fluorescent dye. Inone such method, the single-stranded DNA are separated into fouraliquots at B11, which are used as templates to synthesize DNA withsynthesis terminating at given base types A, G, C or T in a randommanner to obtain a plurality of different molecular weight strands. Thelimited synthesis is obtained by using nucleotides which will terminatesynthesis and is performed in separate containers, one of which has thespecial A nucleotide, another the special G nucleotide, another thespecial C nucleotide and another the special T nucleotide. These specialnucleotides may be dideoxy nucleotides or other nucleotides, includingmarked nucleotides, which terminate synthesis, so that each of the fourbatches are randomly terminated at a different one of the types of basesA, G, C and T. If the nucleotides are marked, such marking may be withfluorescent markers. After being separated by electrophoresis, the bandsare detected by light.

To mark DNA fragments, the template DNA strands are hybridized with aDNA priming oligonucleotide which has a fluorescent marker attached toit. The marker primer is elongated randomly to a selected base type. Inanother embodiment, the priming DNA oligonucleotide is unmarked, butsynthesis is terminated with a fluorescently marked special nucleotide.In either case, the strands are marked. In the preferred embodiment,they are marked with a infrared fluorescent molecule.

To mark the DNA strand, a known infrared dye is modified to provide thedesired wavelengths of maximum absorption and fluorescence. There aremany such dyes such as for example: (1) 3,3'-DiethylthiadicarbocyanineIodide; (2) 3,3'-Diethylthiatricarbocyanine Perchlorate; (3)3,3'-Diethyloxatricarbocyanine Iodide; (4)1,1',3,3,3'-Hexamethylindotricarbocyanine Perchlorate; (5)1,1'-Diethyl-2,2'-dicarbocyanine Iodide; (6)3,3'-Diethylthiadicarbocyanine Iodide; (7)3,3'-Diethyloxatricarbocyanine Iodide; (8)1,1',3,3,3',3'-Hexamethylindotricarbocyanine Perchlorate; (9)1,1',3,3,3',3'-Hexamethylindotricarbocyanine Iodide; and (10)Indocyanine Green.

In the preferred embodiment, the dye has the formula shown in formula 1,with R being --CH₂ --CH₃. This dye is close to having the desiredwavelength of maximum fluorescence and the wavelength of maximumabsorbance may be modified by changing the functional group R. Theunmodified dye may be obtained from Laboratory and Research ProductsDivision, Eastman Kodak Company, Rochester, N.Y. 14650. It is advertisedin the Kodak laser dyes, Kodak publication JJ-169.

The modifications can be made in a manner known in the art. For example,changes occur when different esters are formed replacing the ethylalcohol in the original dye molecule (R equal --CH₂ --CH₃ of formula 1).If different glycol esters are formed, absorption maxima of these newnear infrared dyes shift to the longer wavelengths. Moreover, new dyesmay be synthesized rather than modifying existing dyes in a manner knownin the art.

The absorption maximum is dependent on the distance of the 0 atoms inthe glycol functional group. However, the fluorescence maxima of thesenew near infrared dyes are practically at same wavelength of the dye offormula 1, i.e. 819 nm. This indicates that only the excitation processhas changed, i.e. to what energy level the transition occurs. The lowestvibronic level of first excited state remains unchanged. The absorptionmaxima of several such esters are: (1) ethylene glycol 796 nm(nanometers); (2) 1,3-Propanediol 780 nm; (3) 1,4-Butanediol 754 nm; (4)1,6-Hexanediol 744 nm; (5) Triethylene glycol (#4) 790 nm; and (6)IR-144 (R═CH₂ --CH₃) 742 nm.

The modification to 1,3-propanediol is illustrated in equation 1.

In the preferred embodiment, the fluorescence maximum wavelength isabout 819 nanometers and the detector is adjusted to receive thiswavelength and not others by appropriate filtering. The absorptionmaxima is selected to be different and to correspond to the preferredavailable laser diode emission. For example, in equation 1, R may be anyof the following four groups, depending on the desired wavelength of theemitted light, which are:

(1) --CH₂ --CH₂ --OH for an emission wavelength of 796 nanometers;

(2) --CH₂ --CH₂ --CH₂ --OH for an emission wavelength of 780 nanometers,which is the preferred embodiment;

(3) --CH₂ --CH₂ --CH₂ --CH₂ --CH₂ --CH₂ --OH for an emission wavelengthof 745 nanometers;

(4) --CH₂ --CH₂ --O--CH₂ --CH₂ --O--CH₂ --O--CH₂ --OH for an emissionwavelength of 790 nanometers; and

(5) --CH₂ --CH₂ --SH for an emission wavelength of 810 nanometers.

The preferred and other dyes and methods of using them are more fullydescribed in U.S. patent application Ser. No. 07/763,230 to Middendorfet al ##STR1## entitled, "Sequencing Near Infrared and InfraredFluorescense Fabeled DNA for Detecting Using Laser Diodes" filed Sep.20, 1991, the desclosure of which is incorporated herein by reference.

In each of the embodiments, the dyes may be incorporated in probes andprimers for attachment to oligonucleotides as described in Ruth, JerryL. (1984) DNA 3, 123. The --OH group provides appropriate linkage toconventional probes by reaction with the appropriate reactive group suchas primary amine, carboxylic acid groups and the like but near infrareddyes can be modified to have reactive groups other than the --OH forthis purpose.

To separate the fragments, the marked fragments are each individuallyelectrophoresced through gel in different channels or in differentcolumns. The gel and the field must be uniform although the optional useof one or more reference channels reduces uniformity requirements. Whena single slab is used to migrate several different samples, the channelsmust be kept separate but should be sufficiently close to one another sothat the voltage gradient and temperature are uniform for each channel.Preferably, the pH of the gel for separation is 7-10.

The DNA fragments separate in accordance with their length duringelectrophoresis. Thus, the fastest migrating fraction is the fragmentwhich is synthesized to the first base closest to the priming DNAoligonucleotide and since the channels are separate, it is known whichbase A, G, C or T is the first one in the sequence from the channel.

The next band in time in the gel is the molecule or molecules which isone base longer than the first one since it encompasses both the firstbase and the second one from priming DNA oligonucleotide. Similarly, thethird fragment to form a band during electrophoresis encompasses thefirst three base units and so on.

Because a large number of bases are to be sequenced, there is a largenumber of bands of DNA strands and the number of strands in each band isrelatively low. Thus, the gel and the voltage field must be selected toprovide adequate separation for detection. The gel slab is sufficientlylong such that the more mobile bands near the end of the gel are fullyresolved while the less mobile bands near the entrance end of the gelare unresolved in a continuous process. More specifically, at least 10percent of the bands have been resolved by electrophoresis in the gelwhile the less mobile bands which are near the entrance end of the gelare not fully resolved.

In one embodiment, the bands are scanned by a light source that appliespulsed or chopped light at a repetition rate of 100-15,000 hz. Thefrequency of the light in the pulses is within the optimum absorptionspectrum of the fluorescent marker. The light is sensed using a lock-inamplifier which uses sychronous demodulation to discriminate thefluorescent signal from the DNA strands against the backgroundfluorescent signal, which has a longer time constant. The resultingelectrical signal is amplified and correlated to provide the sequence ofDNA.

In FIG. 8, there is shown a block diagram of another embodiment D10 ofthe invention used for identifying band patterns of DNA strands preparedby such techniques as restriction enzyme cutting or polymerase chainreaction (PCR). The DNA strands are marked by direct labelling offluorescent markers to the strands or by detectingfluorescently-labelled probes hybridized to the separated strands.

Cutting DNA strands with restriction enzymes and then electrophoresingsuch strands results in band patterns known as DNA restriction fragmentlength polymorphisms (RFLP). In restriction fragment lengthpolymorphisms or in the diagnosis of DNA using polymerase chain reaction(PCR), a fingerprint or identification of a single type of DNA isobtained using two identifying components, which are: (1) restrictionenzymes or PCR primers; and (2) marked probes. The use of restrictionfragment length polymorphisms (RFLP) is described generally inBioScience vol. 34, no. 7, pages 410-412 "Linking Diseases to TheirGenes" by Lynn J. Cave.

The first component is utilized in a conventional manner to treatcellular DNA and produce the fragments near or within a gene ofinterest. The restriction enzymes cut only at specific nucleotidesequences, referred to as recognition sites. The PCR primers allowamplification of only those DNA strands having sequences complementaryto such primers.

The second component is used to identify the inherited nucleotidepatterns. To identify the inherited nucleotide patterns, which in somecases indicate a disease or other unique characteristic, patterns areobtained indicating the presence of fragments generated by restrictionenzyme cutting or PCR amplification from a number of cells of a relatedgroup such as of a family. These patterns are compared. An abnormalpattern indicates an abnormal gene and may be identified when correlatedwith the pattern of other members having the same gene pattern in thegroup. Thus, a particular gene representing a disease or abnormality orother feature of interest such as characteristics of a particular strainof plant; or the presence of foreign genetic material; or the like isidentified.

The basic steps of RFLP or of generating fragments by PCR are not bythemselves part of the invention but only the method and apparatusdescribed herein for expediting the obtaining of the patterns byenabling continuous processing in a manner similar to the otherembodiments of this invention. In this process, the DNA fragments areeither fluorescently labelled directly or hybridized with fluorescentlylabelled probes and then electrophoresed in the channel D42. Thefragments are electrophoresed into the sample volume D43, which may, inactuality, be an extension of the gel, where they are detected asdescribed in the embodiments of FIGS. 4 and 7. As they flow through thechannels, the fragments are detected and an identifying indicationrecorded on the recorder 112. The recorder 112 may be a strip chartrecorder, magnetic recorder or any other recorder which indicates thesequence of the fragments for comparison with other patterns against atime base from similar cells to provide an indication of differences.

To provide an improved DNA typing procedure, a prior art radioactivetechnique for digital typing of DNA is modified for use with an opticalon-line scanner. For this purpose, the technique described in theaforementioned article, Jeffreys, A., A. MacLeod, K. Tamaki, D. Neil,and D. Monckton; Mini Satellite Repeat Coding As A Digital Approach ToDNA Typing; Nature 354:204-209, 1991, is modified and used. Themodifications include using a 32D probe which has an extension of thesame sequence as another primer that has an infrared dye attached;sequencing the fragments of different length (sequential typing)according to the above procedures using a denaturing acrylamide gel asthe separation medium; and collecting data in a continuous fashion asdescribed above.

Generally, a DNA locus that varies in sequence from one individual toanother is amplified using PCR and processed to be sequentially typed bythe laser sequencer as described in this application. This can beaccomplished by determining the sequential typing information and usingthe sequential typing information as a code to identify the DNA.Preferably a portion of the locus is amplified or a correspondingsection of bases for each of several localities such as the definedterminal bases of multiple tandem repeats are amplified by PCR to form aplurality of identical strands. The strands are sequentially typed asdescribed above. The sequential typing information is then consideredthe identification code. The typing information is designated in binaryfashion for information coded by units having two differing basesequence structures or in ternary fashion for information coded by unitshaving three differing base sequence structures or the like. The digitalpatterns are compared so that the same code indicates the sameindividuals.

The polymerase chain reaction (PCR) is performed in a standard mannerfor adequate amplification such as for example the procedure describedin U.S. Pat. No. 4,683,202 granted Jul. 28, 1987, to Mullis, thedisclosure of which is incorporated herein by reference. The preferredlocus is D1S8 described in Jeffreys, A. J. Neumann, R. & Wilson, V. Cell60, 473-485 (1990). The hypervariable locus D1S8 (probe MS32) has twoclasses of repeat unit (a-type and t-type) that differ by a single basesubstitution which creates or destroys a HaeIII restriction site.Interspersion patterns of HaeIII⁺ and HaeIII⁻ repeat units are amplifiedby PCR amplification.

In this amplification, two different MVR-specific primers which primeoff either a-type or t-type repeat units are used. Amplification usingone or other primer together with amplimer 32D from a fixed site in theminisatellite flanking DNA generates two complementary sets of productsfrom the ultravariable end of any MS32 allele, from which the MVR mapcan be deduced. To prevent progressive shortening at each PCR cyclebecause of MVR-specific primers priming internally in PCR products, MVRdetection and subsequent amplification were uncoupled by providing eachMVR-specific primer with a 20-nucleotide (nt) 5' extension `TAG` andcarrying out amplifications with a low concentration of one or othertagged primer and high concentrations of 32D and the TAG sequenceitself.

Application of MVR-PCR at limited cycle number to MS32 alleles separatedfrom genomic DNA generated continuous complementary ladders of PCRproducts detectable by fluorescent labels during continuouselectrophoresis. As described above, the primers are labeled withnear-infrared dye for use in sequencing to generate a binary code thatserves as a marker.

In FIG. 9, there is shown another embodiment E10 of sequencing systemhaving a central system 120 and a plurality of remote stations, two ofwhich are shown at 122A and 122B. The remote stations 122A and 122B eachare able to perform the sequencing but some portions of data processingcan only be performed by the central station 120. It may supply data tothe remote stations, such as 122A and 122B, to which it is electricallyconnected and receive data from them. With this arrangement, the centralsequencing system 120 may cooperate with one or more of the remotestations, such as 122A and 122B, for increased capability such asincreased number of channels. Each unit may control the parameters usedin sequencing, such as the electrophoresis potential or the like.

In FIG. 10, there is shown a simplified view of the remote station 122Ahaving a cabinet housing 130, a front cover 132, a liquid crystaldisplay readout 134, a high voltage warning light 136 and a plurality offunction keys 138. In FIG. 10, the remote system 122A is shown closed.However, the front cover 132 may be removed to expose an electrophoresissection. The potential applied across the gel may be set and differentdata readouts may be selected either from the analysis provided withinthe central system 120 (FIG. 9) or values from within the remote station122A using the function key pad 138 and the selected data displayed onthe liquid crystal display readout 134 prior to and/or after selection.

In FIG. 11, there is shown a sectional view of the remote station 122Ataken through section lines 11--11 of FIG. 10 having an electrophoresissection 140, a scanning section 142, an electrophoresis power supply144, a system power supply section 144A, an analog board 146 and adigital board 148. The electrophoresis section 140 is positioned nearthe front of the cabinet and a portion of it is adapted to be scanned bythe scanning section 142 in cooperation with circuitry on the analogboard 146 and the digital board 148. All of the apparatus areelectrically connected to the power supply section 144A for suchoperation.

To separate different DNA fragments into bands, the electrophoresissection 140 includes a gel sandwich 150, an upper buffer assembly 152,support assembly 154, and a lower buffer assembly 151 positioned toenclose the bottom of the gel sandwich 150. In the embodiment of FIG.11, the gel sandwich 150 is held substantially vertically and itstemperature is controlled during operation. Bands are separated byapplying voltage to the upper buffer assembly 152 and lower bufferassembly 151 and scanned by the scanning section 142.

To support the gel sandwich 150, the support assembly 154 includes apair of upper side brackets and lower side brackets 160 and 162 (onlyone of each pair being shown in FIG. 11), a temperature control heatingplate 164, and a plastic spacer, shown at 166A-166C, in FIG. 11. Theentire structure is supported on the support assembly 154 which mountsthe upper and lower side brackets 160 and 162.

The upper and lower side brackets 160 and 162 are shaped to receive thegel sandwich 150 and hold it in place in juxtaposition with the scanningsection 142. The spacer as shown as 166A-166C space the temperaturecontrol heating plate 164 from an apparatus support plate 168 andmaintain it at a constant selected temperature above ambienttemperature. In the preferred embodiment, the temperature is maintainedat 50 degrees Centigrade and should be maintained in a range of 30degrees to 80 degrees.

The scanning section 142 includes a laser diode assembly (not shown inFIG. 11), a microscope assembly 172, a photodiode section 174 and ascanner mounting section 176. The laser diode assembly (not shown inFIG. 11) is positioned at an angle to an opening in the heating plate164 so that light impinges on the gel sandwich 150 to cause fluorescencewith minimum reflection back through the microscope assembly 172.

To receive the fluorescent light, the microscope assembly 172 is focusedon the gel sandwich 150 and transmits fluorescent light emittedtherefrom into the photodiode section 174 which converts it toelectrical signals for transmission to and processing by the analog anddigital boards 146 and 148 which may provide further analysis of data.The scanning section 142 moves along a slot in the apparatus supportplate 168 which is mounted to the scanner mounting section 176 duringthis operation in order to scan across the columns in the gel sandwich150.

The scanner mounting section 176 includes a mounting plate 180, abearing 182, a stepping motor 184, a slidable support 186 and a belt andpully arrangement 185, 188, 188A. The mounting plate 180 is bolted tothe appartus support plate 168 and supports an elongated bearing plate182, a stepping motor 184 and two pulleys 188 and 188A. The elongatedbearing plate 182 extends the length of the gel sandwich 150.

To permit motion of the laser diode assembly (now shown) and microscopeassembly 172 with respect to the gel sandwich 150, the slidable support186 supports the microscope assembly 172 and diode assembly and slidablyrests upon the bearing plate 182. An output shaft 183 of the steppingmotor 184 drives a pulley 188B through pulley 188, belt 185, and pulley188A and the pulley 188B drives a belt (not shown) that is clamped tothe slidable support 186 to move it the length of the gel sandwich 150during scanning by the laser diode and microscope assembly 172 whichrest upon it. The stepping motor 184 under the control of circuitry inthe digital board 148 moves the pulley 188B to move the belt (not shown)and thus cause scanning across the gel sandwich 150.

As shown in this view, the electrophoresis power supply 144 iselectrically connected to buffer in the upper buffer assembly 152through an electrical connector 194 and to the lower buffer assembly 151through a connector not shown in FIG. 11.

The upper buffer assembly 152 includes walls 197 forming a container tohold a buffer solution 195 and a cover 199 formed with a lip to fit overthe walls 197 from the top and containing a downwardly extending flatmember spaced away from the side walls and holding a conductor 211. Theconductor 211 is electrically connected to the source of power throughconnector 194 which is mounted to the top of the cover 199 to permitelectrical energization of the buffer solution 195.

The bottom buffer assembly 151 includes enclosed walls 201 defining acontainer for holding a buffer solution 203 and a cap 205 closing thecontainer 201 and having a downwardly extending portion 213 extendinginto the buffer 203 for supporting a conductor 207 for applying energyto the bottom buffer solution 203. The gel sandwich 150 extendsdownwardly into the buffer solution 203 and upwardly into the buffersolution 195 to permit the electrical contact for electrophoresis.

In FIG. 12, there is shown a sectional view taken through lines 12--12of FIG. 10 showing the electrophoresis section 140, the scanning section142 and the electrophoresis power supply section 144 mounted together toillustrate from a top view the arrangement of the apparatus supportplate 168, the heater plate 164, the gel sandwich 150, a microscopeassembly 172 and a photodiode assembly 174. The heater plate 164 andapparatus support plate 168 have slots running in a horizontal directionorthogonal to the lanes of DNA in the electrophoresis section 140 sizedto receive the ends of a laser diode assembly 170 and the microscopesection 172 for scanning thereof.

To cooperate with the separation and scanning of DNA bands, the gelsandwich 150 includes a front glass plate 200, a gel section 202 and arear glass plate 204 mounted in contact with the heater plate 164 andhaving a section exposed for scanning by the laser diode assembly 170and the microscope assembly 172. The rear glass plate 204 contacts theheater plate 164 and is separated from the front plate 200 by the gelsection 202 within which DNA separation takes place.

To transmit light to the gel sandwich 150, the laser diode assembly 170includes a housing 210, a focusing lens 212, a narrow band pass filter214, a collimating lens 216 and a laser diode 218. The laser diode 218emits infrared or near infrared light which is collimated by the lasercollimating lens 216 and filtered through the narrow band pass infraredfilter 214. This light is focused by the focusing lens 212 onto the gelsandwich 150. Preferably, the point of focus on the gel section 202 ofthe gel sandwich 150 lies along or near the central longitudinal axis ofthe microscope section 172 and the photodiode section 174.

The thickness of the glass plates and the gel, the position of the laserand sensor and their angle of incidence are chosen, taking intoconsideration the refractive index of the gel and glass so that thelight from the laser is absorbed by a maximum number of markers for onechannel. The light from the laser is not directly reflected back becausethe angle of incidence to a normal is equal to the Brewster's angle atthe first interface and is such as to impinge on the markers with fullintensity after refraction but not be reflected by subsequent layers ofthe gel sandwich 150 into the sensor and the sensor views a large numberof markers that fluoresce in a line of sight of substantialconcentration.

To maintain temperature control over the laser diode, the housing 210:(a) is coupled to a heat sink through a thermal electric cooler 220, and(b) encloses the focusing lens 212, narrow band pass filter 214,collimating lens 216 and laser diode 218; and (c) accommodates theelectrical leads for the diode.

To receive and focus light emitted by fluorescent markers from the gelsection 202 in response to the light from the laser diode assembly 170,the microscope assembly 172 includes a collection lens 230, a housing232 and a coupling section 234. The microscope assembly 172 is adaptedto be positioned with its longitudinal axis centered on the collectionlens 230 and aligned with the photodiode section 174 to which it isconnected by the coupling section 234. For this purpose, the housing 232includes a central passageway in which are located one or more opticalfilters with a band pass matching the emission fluorescence of themarked DNA strands along its longitudinal axis from the axis of thecollection lens 230 to the coupling section 234 which transmits light tothe photodiode section 174. With this arrangement, the collection lens230 receives light from the fluorescent material within the gel section202 and collimates the collected light for optical filtering and thentransmission to the photodiode assembly 174.

To generate electrical signals representing the detected fluorescence,the photodiode assembly 174 includes a housing 240 having within it, asthe principal elements of the light sensors, an inlet window 242, afocusing lens 244, a sapphire window 246 and an avalanche photodiode248. To support the avalanche photodiode 248, a detector mounting plate250 is mounted within the housing 240 to support a plate upon which theavalanche photodiode 248 is mounted. The inlet window 242 fits withinthe coupling section 234 to receive light along the longitudinal axis ofthe photodiode assembly 174 from the microscope assembly 172.

Within the housing 240 of the photodiode assembly 174, the sapphirewindow 246 and avalanche photodiode 248 are aligned along the commonaxis of the microscope assembly 172 and the photodiode assembly 174 andfocuses light transmitted by the microscope assembly 172 onto a smallspot on the avalanche photodiode 248 for conversion to electricalsignals. A thermoelectric cooler 252 utilizing the Peltier effect ismounted adjacent to the detector mounting plate 250 to maintain arelatively cool temperature suitable for proper operation of theavalanche photodiode 248.

The lower buffer assembly 151 (FIG. 11) includes outer walls 201 and abottom wall forming a compartment for buffer solution which encloses thebottom of the gel sandwich 150.

As best shown in this view, the stepping motor 184 rotates the belt 185to turn the pulley 188A, which, in turn, rotates pulley 188B. The pulley188B includes a belt 177 extending between it and an idler pulley 179and attached at one location to the slideable support 186 to move thescanning microscope and laser lengthwise along the gel sandwich 150 forscanning purposes. The motor 184 by moving the carriage back and forthaccomplishes scanning of the gel sandwich 150.

In FIG. 13, there is shown a fragmentary perspective view of the gelsandwich 150 and the upper buffer assembly 152 mounted to each othershowing the outer glass plate 200 cut away from the rear glass plate 204to expose the gel section 202 to buffer solution within the upper bufferassembly 152. With this arrangement, samples may be pipetted between theglass plates 200 and 204 and moved downwardly by electrophoresis beyondthe upper buffer assembly 152 and through the gel sandwich 150 to thebottom buffer (not shown in FIG. 13).

In FIG. 14, there is shown a broken away view of the gel sandwich 150illustrating the upper buffer assembly 152 and the lower buffer assembly151 connected to it at each end. As shown in this view, the cover 199includes a connecting post 214 which receives the conductor 211 forconnection to the downwardly extending portion of the cover 199 into thebuffer compartment. Between the glass plates 200 and 204 (FIG. 13) ofthe gel sandwich 150, are a plurality of downwardly extending recesses221 in the gel section 202 (FIG. 13) between the plates. DNA sample ispipetted into these recesses to form channels for electrophoresing tothe lower buffer assembly 151.

To form an electical connection through the gel sandwich 150 from theupper buffer assembly 152 to the lower buffer assembly 151, a conductingpost 216 is connected to the cover 205 of the lower buffer assembly 151for receiving the conductor 207 which extends downwardly to thedownwardly extended plate 213 and into the buffer solution.

In FIG. 15, there is shown a block diagram of the circuitry used tocontrol the remote station 122A of the embodiment of FIG. 11 having acontrol, correlation and readout section 250, the scanner drive 176, themotor assembly 184 for moving the scanner drive 176, and the sensingconfiguration 252. The sensing configuration 252 includes the laserassembly 170 and the sensor assembly 174 which receives signals, removessome noise, and transmits the signals for display and read out in thecontrol, correlation and read out section 250 while the scanner drive176 and motor for the scanner drive 184 receive signals from thecontrol, correlation and read out section 250 to control the motion ofthe sensor back and forth across the gel sandwich. This overallconfiguration is not part of the invention of this application exceptinsofar as it cooperates with the sensing configuration 252 to scan theDNA and determine its sequence in accordance with the embodiments ofFIGS. 9-14.

To drive the sensor 174 from position to position, the motor assembly184 includes a stepper motor 254 and a motor driver 256. The motordriver 256 receives signals from the control correlation and read-outsection 250 and actuates the stepper motor 254 to drive the scannerdrive 176. The scanner drive 176 is mechanically coupled to a steppingmotor 254 through a belt and pulley arrangement for movement back andforth to sense the electrophoresis channels on the gel sandwich 150(FIG. 12). The stepping motor 254 and driver circuitry 256 areconvention and not themselves part of the invention.

The control, correlation and read out system 250 includes a computerwhich may be any standard microprocessor 260, a television display orcathode ray tube display 262 and a printer 264 for displaying andprinting the results of the scans.

To sense data, the sensing configuration 252 includes in addition to thelaser 170 and the sensor 174, a chopper circuit 270, a sensor powersupply 272, a preamplifier 274, a lock-in amplifier 276, a 6-pole filter278, a 12-bit analogue digital converter interface circuit 280 and alaser power supply 282.

The sensor 174 receives light from the laser 170 after it impinges uponthe gel sandwich 150 (FIG. 12) and transmits the signals throughpreamplifier 274 to the lock-in amplifier 276. The sensor receivessignals from the sensor power supply 272. The chopper circuit 270provides pulses at synchronized frequencies to the lock-in amplifier276.

The laser 170 receives power from the power supply 282 which iscontrolled by the chopper circuit 270 so that the signal from the laseris in synchronism with the signal applied to the lock-in amplifier 276so that the output from the lock-in amplifier 276 to the 6-pole filter278 discriminates against unwanted signal frequencies. This signal isconverted to a digital signal in the 12-bit analogue to digitalconverter 280 which serves as an interface to the computer 260.

With this arrangement, the scanning rate may be set to discriminateagainst noise and the synchronized demodulation from the chopper controlfurther reduces noise, particularly discriminating against the naturalfluorescense of the glass in the gel sandwich 150 (FIGS. 11 and 12).

From the above summary, it can be understood that the sequencingtechniques of this invention have several advantages, such as: (1) theytake advantage of resolution over time, as opposed to space; (2) theyare continuous; (3) they are automatic; (4) they are capable ofsequencing or identifying markers in relatively long strands includingstrands of more than 100 bases; and (5) they are relatively economicaland easy to use.

While in the preferred embodiment, a single emission frequency is usedin the infrared region in each channel and for all of A, T, G and Cterminated strands with the channel location identifying the terminatingbase type, multiple fluorescent markers can be used with the wavelengthbeing used to identify the base type. In such an embodiment, an opticalmeans detects a plurality of wavelengths and the computer correlatesintensity data, corresponding lanes and corresponding wavelengths.

Although a preferred embodiment of the invention has been described withsome particularity, many modifications and variations are possible inthe preferred embodiment within the light of the above description.Accordingly, within the scope of the appended claims, the invention maybe practiced other than as specifically described.

What is claimed is:
 1. A method of typing DNA, comprising:amplifying atleast certain bases on a selected locus of the DNA, wherein the selectedlocus has variability in its sequence using PCR; flourescently markingthe at least some of the amplified bases; applying DNA fragments atleast at one of a plurality of locations for electrophoresing in atleast one of a plurality of channels through a gel electrophoresis slab;establishing electrical potential across said gel electrophoresis slabwherein the DNA fragments are resolved in accordance with the size ofDNA fragments in said gel electrophoresis slab into fluorescently markedDNA bands; scanning the separated fragments photoelectrically with alaser and a sensor wherein the laser scans with scanning light at ascanning light frequency within the absorbance spectrum of saidfluorescently marked DNA fragments; sensing light at the emissionfrequency of the marked DNA fragments; and comparing the sensed labelsfrom the patterns, wherein individuals are identified.
 2. A method ofDNA sequencing comprising the steps of:applying opposite polarityelectrical potentials to a first and at least second buffer; applyingfluorescently marked bases to a plurality of channels of gel, wherebysaid fluorescently marked DNA are electrophoresced along said gel sothat the bands of the more mobile strands in at least one channel isfully resolved while some of the less mobile strands to be later formedinto bands are unresolved in a continuous process; scanning across saidchannels with light emitted from a diode laser; detecting fluorescentlight emitted by said fluorescently marked bases, whereby the timesequence of separated bands may be obtained; said step of detectingincluding the step of scanning across said channels with a microscopefocused on the bands receiving said light; sensing light at the emissionfrequency of the marked DNA; comparing the sensed bases from thepatterns, wherein individuals are identified.
 3. A method of DNAsequencing comprising the steps of:applying opposite polarity electricalpotentials to a first and at least second buffer; applying fluorescentlymarked alleles to a plurality of channels of gel, whereby saidfluorescently marked alleles are electrophoresced along said gel so thatthe bands of the more mobile strands in at least one channel are fullyresolved while some of the less mobile strands to be later formed intobands are unresolved in a continuous process; scanning across saidchannels with light emitted from a laser; and detecting fluorescentlight emitted by said fluorescently marked, alleles whereby the timesequence of separated bands of alleles may be obtained wherein the lightfrom said laser is in a band of wavelenghts of light incorporating atleast the near infrared and infrared regions and said detector respondsto light in a band of wavelenghts of light including at least said nearinfrared and infrared regions; and comparing the sensed bases from thepatterns, wherein individuals are identified.
 4. A method according toclaim 2 wherein said microscope and diode laser are moved togetheracross said channels to perform said scanning.
 5. A method according toclaim 2 wherein the light from the diode laser is scanned at an anglechosen to impinge on the bands with full intensity after refraction butnot be reflected into the microscope.
 6. A method according to claim 5wherein the light from the diode laser is scanned at an angle to thesurface being scanned equal to the Brewster's angle.
 7. DNA sequencingapparatus comprising:gel electrophoresis means; one end of said gelelectrophoresis means communicating with a buffer solution; at least oneother side of said gel electrophoresis means communicating with a secondbuffer solution; means for applying opposite polarity electricalpotentials to said first and at least second buffer; means for receivinga plurality of fluorescently marked DNA bases from an individual's DNAin a plurality of channels of said gel electrophoresis means, wherebysaid fluorescently marked DNA is electrophoresed along said gelelectrophoresis means so that the bands of the more mobile strands in atleast one channel are fully resolved while some of the less mobilestrands to be later formed into bands are unresolved in a continuousprocess; means mounted to move with respect to said gel for scanningacross said channels with light emitted from a diode laser; said meansmounted to move including a carriage, said diode laser and a means fordetecting fluorescent light emitted by said fluorescently marked bases,whereby the time sequence of separated bands may be obtained to providea sensed pattern indicative of an individual; said means for detectingincluding means for scanning across said channels with a microscopemounted to said carriage and focused on the bands receiving said light;and means for comparing the sensed patterns of DNA with otherinformation to identify the individual.
 8. DNA sequencing apparatuscomprising:gel electrophoresis means; one end of said gelelectrophoresis means communicating with a buffer solution; at least oneother side of said gel electrophoresis means communicating with a secondbuffer solution; means for applying opposite polarity electricalpotentials to said first and at least second buffer solutions; means forreceiving fluorescently marked alleles in a plurality of channels ofsaid gel electrophoresis means, whereby said fluorescently marked DNA iselectrophoresed along said gel electrophoresis means so that the bandsof the more mobile strands in at least one channel are fully resolvedwhile some of the less mobile strands to be later formed into bands areunresolved in a continuous process; means for scanning across saidchannels with light emitted from a laser; means for detectingfluorescent light entitled by said fluorescently marked strands, wherebythe time sequence of separated bands may be obtained; said means forscanning emitting including means for scanning with light from saidlaser in a band of wavelenghts of light incorporating at least the nearinfrared and infrared regions; said detector being responsive to lightin a band of wavelengths including at least said near infrared andinfrared regions; means for recording sensed patterns of bands, whereinalleles are detected from the patterns so that individuals areidentified.
 9. DNA sequencing apparatus according to claim 8 furtherincluding means for moving said microscope and diode laser togetheracross said channels to perform said scanning.
 10. DNA sequencingapparatus according to claim 8 wherein the light from the diode laser isscanned at an angle chosen to impinge on the bands with full intensityafter refraction but not be reflected into the microscope.
 11. DNAsequencing apparatus according to claim 10 wherein the light from thediode laser is scanned at an angle to the surface being scanned equal tothe Brewster's angle.